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Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

Klein SR, Jiang H, Hossain MB, Fan X, Gumin J, Dong A, Alonso MM, Gomez-Manzano C, Fueyo J - PLoS ONE (2016)

Bottom Line: We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation.Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner.However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-Oncology, Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

No MeSH data available.


Related in: MedlinePlus

Autophagy is associated with viral antigen presentation.(a) Mock- or Δ24RGD-infected wild-type MEFs were pre-incubated with antibodies against MHC class I, MCH class II, or IgG isotope and then co-cultured with splenocytes obtained from Δ24RGD-infected mice. The fold-change of IFN-γ levels (pg/mL) in the co-culture media is displayed relative to control and shown as mean ± SD. ** P < 0.01 (unpaired, two-tailed Student t-test). (b) Wild-type MEF cells were transfected with a pool of siNC, siMHCI, siMHCII, or combined equal amounts of siMHC I and siMHC II for 48 h and then infected with the Δ24RGD adenovirus at an MOI of 10 for an additional 48 h. Whole-cell lysates were analyzed by using anti-MHC class I and anti-MHC class II antibodies. The effect of each siRNA treatment on the protein level of MHC molecules is illustrated. (c) Wild-type MEFs were transfected with a pool of siNC, siMHCI, siMHCII, or combined equal amounts of siMHCI and siMHCII for 48 hours, and then infected with the Δ24RGD adenovirus at an MOI of 10 for an additional 48 h. Then, cells were stained for detection of adenoviral antigens. Propidium iodide was used to assess cell viability. Data are shown as percentage of positive cells (mean ± SD). The decrease in the percent of positive adenovirus-infected, siMHCII-transfected cells compared with adenovirus-infected, siNC-transfected cells was statistically significant. ** P < 0.01 (unpaired, two-tailed Student’s t-test).
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pone.0153814.g002: Autophagy is associated with viral antigen presentation.(a) Mock- or Δ24RGD-infected wild-type MEFs were pre-incubated with antibodies against MHC class I, MCH class II, or IgG isotope and then co-cultured with splenocytes obtained from Δ24RGD-infected mice. The fold-change of IFN-γ levels (pg/mL) in the co-culture media is displayed relative to control and shown as mean ± SD. ** P < 0.01 (unpaired, two-tailed Student t-test). (b) Wild-type MEF cells were transfected with a pool of siNC, siMHCI, siMHCII, or combined equal amounts of siMHC I and siMHC II for 48 h and then infected with the Δ24RGD adenovirus at an MOI of 10 for an additional 48 h. Whole-cell lysates were analyzed by using anti-MHC class I and anti-MHC class II antibodies. The effect of each siRNA treatment on the protein level of MHC molecules is illustrated. (c) Wild-type MEFs were transfected with a pool of siNC, siMHCI, siMHCII, or combined equal amounts of siMHCI and siMHCII for 48 hours, and then infected with the Δ24RGD adenovirus at an MOI of 10 for an additional 48 h. Then, cells were stained for detection of adenoviral antigens. Propidium iodide was used to assess cell viability. Data are shown as percentage of positive cells (mean ± SD). The decrease in the percent of positive adenovirus-infected, siMHCII-transfected cells compared with adenovirus-infected, siNC-transfected cells was statistically significant. ** P < 0.01 (unpaired, two-tailed Student’s t-test).

Mentions: Autophagy is involved in the processing of peptides for presentation mainly via MHC class II molecules,[10, 11] thus we decided to determine whether antibody-based blockade of MHC class II prevented the recognition of infected cells by the adenovirus-primed splenocytes. To this end, we quantified the IFN-γ production in co-cultures of adenovirus-primed splenocytes with virus-infected MEFs pre-incubated with blocking antibodies for MHC class I or II. We observed that cells treated with antibodies against MHC class II proteins showed significant inhibition of IFN-γ production compared with cells treated with antibodies against MHC class I proteins or IgG-treated controls (Fig 2A). To confirm these data we challenged the presentation of adenovirus antigens by using specific siMHC class II (Fig 2B). We observed that down-modulation of MHC class II mRNA resulted in the decrease in the number of cells presenting adenoviral epitopes (Fig 2C). Indicating the predominant role of the MHC class II in the process, showed that the down-modulation of MHC class I had much less effect on the presentation of adenovirus-derived antigens than did the down-regulation of MHC class II. These data indicate that adenovirus-derived antigens are predominantly presented via MHC class II, the predominant pathway of epitope presentation for proteins processed via autophagy.[10, 11]


Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

Klein SR, Jiang H, Hossain MB, Fan X, Gumin J, Dong A, Alonso MM, Gomez-Manzano C, Fueyo J - PLoS ONE (2016)

Autophagy is associated with viral antigen presentation.(a) Mock- or Δ24RGD-infected wild-type MEFs were pre-incubated with antibodies against MHC class I, MCH class II, or IgG isotope and then co-cultured with splenocytes obtained from Δ24RGD-infected mice. The fold-change of IFN-γ levels (pg/mL) in the co-culture media is displayed relative to control and shown as mean ± SD. ** P < 0.01 (unpaired, two-tailed Student t-test). (b) Wild-type MEF cells were transfected with a pool of siNC, siMHCI, siMHCII, or combined equal amounts of siMHC I and siMHC II for 48 h and then infected with the Δ24RGD adenovirus at an MOI of 10 for an additional 48 h. Whole-cell lysates were analyzed by using anti-MHC class I and anti-MHC class II antibodies. The effect of each siRNA treatment on the protein level of MHC molecules is illustrated. (c) Wild-type MEFs were transfected with a pool of siNC, siMHCI, siMHCII, or combined equal amounts of siMHCI and siMHCII for 48 hours, and then infected with the Δ24RGD adenovirus at an MOI of 10 for an additional 48 h. Then, cells were stained for detection of adenoviral antigens. Propidium iodide was used to assess cell viability. Data are shown as percentage of positive cells (mean ± SD). The decrease in the percent of positive adenovirus-infected, siMHCII-transfected cells compared with adenovirus-infected, siNC-transfected cells was statistically significant. ** P < 0.01 (unpaired, two-tailed Student’s t-test).
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pone.0153814.g002: Autophagy is associated with viral antigen presentation.(a) Mock- or Δ24RGD-infected wild-type MEFs were pre-incubated with antibodies against MHC class I, MCH class II, or IgG isotope and then co-cultured with splenocytes obtained from Δ24RGD-infected mice. The fold-change of IFN-γ levels (pg/mL) in the co-culture media is displayed relative to control and shown as mean ± SD. ** P < 0.01 (unpaired, two-tailed Student t-test). (b) Wild-type MEF cells were transfected with a pool of siNC, siMHCI, siMHCII, or combined equal amounts of siMHC I and siMHC II for 48 h and then infected with the Δ24RGD adenovirus at an MOI of 10 for an additional 48 h. Whole-cell lysates were analyzed by using anti-MHC class I and anti-MHC class II antibodies. The effect of each siRNA treatment on the protein level of MHC molecules is illustrated. (c) Wild-type MEFs were transfected with a pool of siNC, siMHCI, siMHCII, or combined equal amounts of siMHCI and siMHCII for 48 hours, and then infected with the Δ24RGD adenovirus at an MOI of 10 for an additional 48 h. Then, cells were stained for detection of adenoviral antigens. Propidium iodide was used to assess cell viability. Data are shown as percentage of positive cells (mean ± SD). The decrease in the percent of positive adenovirus-infected, siMHCII-transfected cells compared with adenovirus-infected, siNC-transfected cells was statistically significant. ** P < 0.01 (unpaired, two-tailed Student’s t-test).
Mentions: Autophagy is involved in the processing of peptides for presentation mainly via MHC class II molecules,[10, 11] thus we decided to determine whether antibody-based blockade of MHC class II prevented the recognition of infected cells by the adenovirus-primed splenocytes. To this end, we quantified the IFN-γ production in co-cultures of adenovirus-primed splenocytes with virus-infected MEFs pre-incubated with blocking antibodies for MHC class I or II. We observed that cells treated with antibodies against MHC class II proteins showed significant inhibition of IFN-γ production compared with cells treated with antibodies against MHC class I proteins or IgG-treated controls (Fig 2A). To confirm these data we challenged the presentation of adenovirus antigens by using specific siMHC class II (Fig 2B). We observed that down-modulation of MHC class II mRNA resulted in the decrease in the number of cells presenting adenoviral epitopes (Fig 2C). Indicating the predominant role of the MHC class II in the process, showed that the down-modulation of MHC class I had much less effect on the presentation of adenovirus-derived antigens than did the down-regulation of MHC class II. These data indicate that adenovirus-derived antigens are predominantly presented via MHC class II, the predominant pathway of epitope presentation for proteins processed via autophagy.[10, 11]

Bottom Line: We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation.Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner.However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-Oncology, Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

No MeSH data available.


Related in: MedlinePlus